Abstract Genital herpes is a sexually transmitted disease primarily caused by herpes simplex virus type 2 (HSV-2). An estimated 20% of the population in the US is infected with HSV-2. Adding to the problems and diseases afflicted by HSV-2 infections, cases of genital infections by HSV-1 have gone up significantly in the past few decades. This creates a complex health issue since much higher percentages of adults may harbor HSV-1. HSV belongs to a subfamily of neurotropic herpesviruses that establish latency in sensory neurons and cause lifelong recurrent infections. Current frontline treatments against genital herpes include acyclovir (ACV) and its analogs. While these have proven effective over the past few decades, the limitations associated with these antivirals such as: a) increasing incidences of drug resistance especially among immunocompromised patients, b) limited efficacy as topical formulations, and c) higher cost of treatment and side effects associated with long- term systemic treatments, restrict their use and underscore the need for new and improved treatment options. The focus of this study is to evaluate the efficacy of a small molecule, Iazovir (IZV), as a viable alternative treatment for genital herpes. We have strong and supportive preliminary data to develop IZV as a brand new class of highly effective antivirals. Two specific aims are proposed that will establish the efficacy and provide the molecular mechanism behind the antiviral action of IZV. Aim 1 will focus on determining the mechanism of antiviral action by IZV. Based on our interesting preliminary results that IZV reduces both HSV genome and transcript levels, we hypothesize that IZV can: (i) block transcription of HSV-2 genomes by TANK binding kinase 1 (TBK1)-mediated modulation of NF-?B activation and (ii) inhibit HSV-2 replication through its suppressive activity on nuclear mitotic apparatus (NuMA). Multiple experiments are planned to test our hypothesis. In parallel, IZV resistant HSV-2 mutants will be generated to provide an unbiased and deeper understanding of the mechanisms that govern the antiviral potential of IZV. Aim 2 will use mouse models of genital HSV-2 infection to determine the in vivo preclinical efficacy of IZV treatment as a topical and oral antiviral therapy. Experiments will also be undertaken to demonstrate IZV?s high efficacy as an oral treatment against murine genital infections caused by HSV-1. We will also determine the acute and chronic toxicities induced by IZV treatment in order to fully characterize its pre-clinical efficacy. Successful completion of our studies will establish IZV as a new class of HSV antivirals ready for future clinical trials and studies.